Bottom Line:
The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion.Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation.The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.

Affiliation: Departments of Pathology and Cell Biology, University of South Florida Morsani College of Medicine, Tampa, FL 33612.

ABSTRACTFe65 is a brain-enriched adaptor protein known for its role in the action of the Aβ amyloid precursor protein in neuronal cells and Alzheimer's disease, but little is known about its functions in cancer cells. The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion. Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation. The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.

f5: Tip60 binds to and acetylates cortactin through Fe65 to inhibit cell motility.(A), Inducible Fe65 stable knockdown clones of 293T cells were transfected with 1.5 μg Flag-cortactin and 1.5 μg of HA-Tip60 and treated with DMSO or 1 μg/mL doxycycline as shown. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. (B), Cells were transfected as in panel A and treated with DMSO, 300 ng/mL TSA and/or 1 μg/mL doxycycline. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. Acetyl-lysine (Ac-K) antibody is an antibody that specifically recognizes lysine-acetylated proteins. (C) and (D), 293T cells were transfected with HA-Tip60 together with control or Fe65 siRNA. 36 h post transfections, 5 × 105 transfected cells were plated into the inserts for trans-well assays. Western blots were performed to show Fe65 knockdown and β-actin blots were included as loading controls (panel C). Cells were fixed and photographed 24 h after plating (panel D). (E) and (F), Quantitative data were presented as bar graphs and data statistics performed as described in Fig. 1.

Mentions:
As mentioned earlier, Tip60 is HAT known to interact with the PTB1 domain of Fe6519. It is thus reasonable to speculate that Tip60 may work through Fe65 to acetylate cortactin and suppress cell migration and invasion. To test this concept, we engineered 293T cells in which Fe65 knockdown is inducible and examined the interaction between co-transfected Flag-cortactin and HA-Tip60 in co-immunoprecipitation assays. As shown in Fig. 5A, Flag beads co-precipitated Tip60 and cortactin in co-transfected cells but not in cells transfected with HA-Tip60 or Flag-cortactin alone, showing a true binding of Tip60 to cortactin. More importantly, doxycycline treatment, which reduced the expression of Fe65 protein as expected due to induced expression of Fe65 shRNA (Fig. 5A), decreased the amount of Tip60 co-precipitated with cortactin (Fig. 5A). The data suggest that Fe65 is the adaptor that mediates the binding of Tip60 to cortactin.

f5: Tip60 binds to and acetylates cortactin through Fe65 to inhibit cell motility.(A), Inducible Fe65 stable knockdown clones of 293T cells were transfected with 1.5 μg Flag-cortactin and 1.5 μg of HA-Tip60 and treated with DMSO or 1 μg/mL doxycycline as shown. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. (B), Cells were transfected as in panel A and treated with DMSO, 300 ng/mL TSA and/or 1 μg/mL doxycycline. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. Acetyl-lysine (Ac-K) antibody is an antibody that specifically recognizes lysine-acetylated proteins. (C) and (D), 293T cells were transfected with HA-Tip60 together with control or Fe65 siRNA. 36 h post transfections, 5 × 105 transfected cells were plated into the inserts for trans-well assays. Western blots were performed to show Fe65 knockdown and β-actin blots were included as loading controls (panel C). Cells were fixed and photographed 24 h after plating (panel D). (E) and (F), Quantitative data were presented as bar graphs and data statistics performed as described in Fig. 1.

Mentions:
As mentioned earlier, Tip60 is HAT known to interact with the PTB1 domain of Fe6519. It is thus reasonable to speculate that Tip60 may work through Fe65 to acetylate cortactin and suppress cell migration and invasion. To test this concept, we engineered 293T cells in which Fe65 knockdown is inducible and examined the interaction between co-transfected Flag-cortactin and HA-Tip60 in co-immunoprecipitation assays. As shown in Fig. 5A, Flag beads co-precipitated Tip60 and cortactin in co-transfected cells but not in cells transfected with HA-Tip60 or Flag-cortactin alone, showing a true binding of Tip60 to cortactin. More importantly, doxycycline treatment, which reduced the expression of Fe65 protein as expected due to induced expression of Fe65 shRNA (Fig. 5A), decreased the amount of Tip60 co-precipitated with cortactin (Fig. 5A). The data suggest that Fe65 is the adaptor that mediates the binding of Tip60 to cortactin.

Bottom Line:
The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion.Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation.The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.

Affiliation:
Departments of Pathology and Cell Biology, University of South Florida Morsani College of Medicine, Tampa, FL 33612.

ABSTRACTFe65 is a brain-enriched adaptor protein known for its role in the action of the Aβ amyloid precursor protein in neuronal cells and Alzheimer's disease, but little is known about its functions in cancer cells. The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion. Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation. The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.